The cultivation of agricultural crop plants serves mainly for the production of foodstuffs for humans and feedingstuffs for animals. The last 25 years have seen pronounced yield increases in crop production. This was the result of a good combination of altered production techniques, newly developed varieties, fertilization and, last but not least, increased crop protection. In the light of an ever increasing world population, safeguarding food production gains increasingly in importance. It has been estimated that 7 billion people will inhabit Earth in 2010. To feed all these people, without the proportion of malnourished people increasing, food production would have to be increased by 60% (Entrup N. L. et al., Lehrbuch des Pflanzenbaues [Textbook of crop production], Thomas Mann Verlag, Gelsenkirchen, 2000). Efficient crop protection is a decisive factor in this context. Monocultures in particular, which are the rule nowadays, are highly susceptible to an epidemic-like spreading of diseases. The result are markedly reduced yields. To date, the pathogenic organisms have been controlled mainly by using pesticides. Nowadays, in contrast, the possibility of directly modifying the genetic disposition of a plant or pathogen is open to man.
Fungi are distributed worldwide so they may form a heterogeneous group with a range of species. They are eukaryotes, do not contain chlorophyll and are therefore heterotrophic. Hence, they rely on external carbon sources which they tap as parasites, saprophytes or symbionts. Saprophytes live exclusively on dead plant material. Parasitic fungi feed on live tissue and must have concluded their development before the plant has died. Facultative parasites can feed both on live and on dead tissue. Symbionts, such as mycorrhiza, live in close association with the plants. Fungi have one or more nuclei per cell and are homokaryotic or heterokaryotic. Fungi have a firm cell wall during at least one stage in their life history. This cell wall usually consists of chitin or, in some cases such as the Oomycota, of cellulose. The vegetative part of the fungus (thallus) is usually haploid, in rare cases diploid. The thallus of lower fungi (Myxomycota, inter alia) consists of ameboidal cells or plasmodia (naked, polynuclear protoplasma). Eumycota have budding cells, as in the case of yeasts (for example Saccharomyces cerevisiae), or form a mycelium which consists of threadlike hyphae. As the result of hyphal aggregation, specific organs for propagation (fruiting bodies) or for surviving unfavorable environmental conditions (sclerotia) may be formed. Propagation and multiplication is usually by way of spores; asexually by means of conidia, uredospores, sporarigiospores, chlamydospores and zoospores, and sexually with oospores, ascospores, zygospores and basidiospores.
Approximately 100 000 different fungal species are known to date. Among these, however, only 5% are plant pathogens. The Basidiomycota are a division of the true fungi which are characterized by the development of a particular structure, the basidium, on which the basidiospores mature. The Basidiomycota also include the generally known mushrooms. The Basidiomycota are predominantly heterothallic and self-sterile. Mating occurs by somatogamy. During somatogamy, two compatible, haploid, mononuclear mycelia or sporidia coalesce. The resulting dikaryotic mycelium constitutes the dominant phase of the life cycle over a prolonged period. The only two phytopathogenic genera of the Basidiomycota are the smuts (Ustilages) and the rusts (Uredinales). Smuts only attack angiosperms and use to be of great economical importance. Nowadays they are controlled successfully by suitable active substances and tight seed control. The rusts are still somewhat more important nowadays. They are biotrophic and can have a complicated development cycle with up to five different spores stages (spermatium, aecidiospore, uredospore, teleutospore and basidiospore). Rusts which develop all spore stages are referred to as macrocyctic rusts. If some stages are absent, these rusts are referred to as being macrocyclic. “Imperfect rusts” lack the basidiospores. Some rusts change their hosts during their development. These are referred to as heteroecious. Host alternation can be linked to nuclear-phase alternation. In contrast, autoecious rusts complete all of their development on one host. A traditional example of a macrocyclic heteroecious rust is black rust of cereals, Puccinia graminis. P. graminis, in its dikaryotic stage, attacks predominantly wheat. The haplont is pathogenic to barberry (Börner H., Pflanzenkrankheiten and Pflanzenschutz [Plant disease and plant protection], Ulmer Verlag Stuttgart, 1997; Sitte P. et al., Strasburger—Lehrbuch der Botanik [Textbook of Botany], Gustav Fischer Verlag, Stuttgart, 1998; Entrup N. L. et al., Lehrbuch des Pflanzenbaues [Textbook of crop production], Thomas Mann Verlag, Gelsenkirchen, 2000).
During the infection of plants by pathogenic fungi, different phases are usually observed. The first phases of the interaction between phytopathogenic fungi and their potential host plants are decisive for the colonization of the plant by the fungus. During the first stage of the infection, the spores become attached to the surface of the plants, germinate, and the fungus penetrates the plant. The attachment of the spores requires either an active metabolism, which is the case in Colletotrichum graminicola, or it is passive, as is the case with Magnaporthe grisea. In the latter case, moisture leads to the secretion of a “mucilage adhesive”, by means of which the spore attaches (Howard R. J. et al., Annu. Rev. Microbiol. 50, 491 (1996)). Spore germination is induced either by unspecific inductors such as water, nutrients, ethylene or, more rarely, as is the case in Phyllosticta ampelicida, by hydrophobic surfaces (Kuo K. et al., Fungal Genet. Biol. 20, 18 (1996)). Some fungi develop two germ tubes, as is the case in powdery mildew cereals, Blumeria graminis, while other fungi develop only one germ tube (Green J. R. et al., The powdery mildews, a comprehensive treatise; The formation and function of infection and feeding structures, APS Press, 2002). Fungi may penetrate the plant via existing ports such as stomata, lenticels, hydatodes and wounds, or else they penetrate the plant epidermis directly as the result of the mechanical force and with the aid of cell-wall-digesting enzymes. Specific infection structures are developed for penetration of the plant. These pressure organs are referred to as appressoria and allow the fungus to build up a high pressure above a discrete point. It is estimated that appressorium of M. grisea reaches a pressure of 80 bar (Howard R. J. et al., Annu. Rev. Microbial. 50, 491 (1996)). Most rusts, in contrast, penetrate the plant via the stomata. The soya rust Phakopsora pachyrhizi directly penetrates the plant epidermis and therefore resembles powdery mildew of cereals, B. graminis, in its penetration behavior (Koch E. et al., Phytopath. p. 106, 302 (1983); Tucker S. L. et al., Annu. Rev. Phytopathol. 39, 385 (2001); Green J. R. et al., The powdery mildews, a comprehensive treatise; The formation and function of infection and feeding structures, APS Press, 2002).
Phytopathogenic fungi do not always colonize all of the plant; in contrast, sometimes it is only specific areas or tissues which are colonized. Following the successful invasion of the plant, phytopathogenic fungi follow different nutritional strategies. Pertotrophic or necrotrophic pathogens kill the host cells by means of extracellular enzymes or toxins and feed by degrading the dead cells. Some genera with pertotrophic nutrition are the Fusaria sp., Alternaria sp. and Cochliobolus. Most fungi use this feeding strategy. The biotrophic phytopathogenic fungi, such as mildew and many rusts, depend, for their nutrition, on the metabolism of live cells. An intermediate position is occupied by the hemibiotrophic pathogenic fungi, which include the genera Phythophtora and Peronospora. Most of these are biotrophs at the beginning of their development and only change over to a pertotrophic lifestyle during the later stages of their development (Prell H. H., Interaktionen von Pflanzen and phytopathogenen Pilzen [Interactions between plants and phytopathogenic fungi], Gustav Fischer Verlag, Jena, 1996). The plants have developed defense mechanisms to avoid infection. Another intermediate position is occupied for example by soybean rust, which penetrates the epidermis directly, whereupon the penetrated cell becomes necrotic; after the penetration, the fungus changes over to an obligatory-biotrophic lifestyle. The subgroup of the biotrophic fungal pathogens which follows essentially such an infection strategy will, for the purposes of the present description, be referred to as being “heminecrotrophic”.
It must be emphasized that plants, during their development, are exposed to constant attack by a large number of phytopathogenic organisms. Nevertheless, colonization of the plant by phytopathogenic organisms is the exception rather than the rule. Before a pathogen can attack the plant, it has to overcome a series of barriers. Frequently, the pathogen has developed specific pathogenicity factors which are adapted to the plant, known as virulence factors, in order to overcome these barriers. In such a case, the plant becomes a host plant for the pathogen, i.e. the latter is virulent on the plant. There is a basic compatibility between the plant and the pathogen. This means that the physiological and biochemical prerequisites which are required for the colonization are already in existence or have been produced (Prell H. H., Interaktionen von Pflanzen und phytopathogenen Pilzen [Interactions between plants and phytopathogenic fungi], Gustav Fischer Verlag, Jena, 1996). In the case of compatible interaction, the pathogen develops at the expense of the plant, thus causing the formation of disease symptoms such as wilting, necroses and chloroses. However, if basic compatibility exists, the plant can still defend itself against the pathogen when a resistance mutation has taken place in the plant. The resistance of the plant, thus acquired, is referred to as host resistance. It is only directed against a certain individual pathogen and can be overcome readily by the latter. The pathogens in question are mostly, but not always, biotrophic pathogens. Host resistance can be subdivided into non-race-specific horizontal resistances which, in most cases, involves several genes, and race-specific vertical resistance. The latter is only effective against certain, individual races of a pathogen, while the plant defends itself a priori against most pathogens. This phenomenon is referred to as basic incompatibility or non-host resistance. In contrast to host resistance, non-host resistance is based on a series of causes and not on individual genes. Firstly, the pathogen may lack the necessary pathogenicity factors, or else the plant is capable of recognizing, and successfully defending itself, against the pathogen. Another term which is important in particular for agriculture is tolerance. A plant is tolerant to a pathogen when it can be attacked, but the attack does not lead to the development of disease systems and yield reduction (Prell H. H., Interaktionen von Pflanzen und phytopathogenen Pilzen [Interactions between plants and phytopathogenic fungi], Gustav Fischer Verlag, Jena, 1996). For the purposes of the description of the present invention, generating, or increasing, a resistance is to comprise an increase or generation of any type of resistance, but also of tolerance, i.e. in particular all cases of an increased tolerance or resistance which lead to reduced yield losses as caused by the pathogen.
In connection with resistance responses of plants, the term resistance factors includes structures, substances and processes which prevent or inhibit attack of the plant by potential pathogens. If the resistance factors are already constitutively present in the plant, they are referred to as pre-formed resistance factors. Induced resistance factors are only formed when a recognition response between the plant and the potential pathogen has taken place. Recognition can be described as a signal/sensor response (elicitor/receptor model, Keen N. T. et al., Phytopathology 62, 768 (1972)). The signal are plant substances or substances produced by the pathogen, known as elicitors, which bind to a sensor or receptor which is specific for the elicitor in question. This binding triggers one or more effectors, which may be, for example, signal transduction chains and induce the resistance response. A whole series of substances act as elicitors. These include proteins, glycoproteins, glucans and lipids (Garcia Brugger et al., MPMI 19, 711 (2006)). Plant cell wall degradation products which are released by enzymes of the pathogen or else by wounding of the plant may also induce a resistance response. In this context, an avirulence factor of the pathogen and the corresponding resistance gene of the plant is frequently mentioned (“Gene-for-gene hypothesis”, nor, J. Agric. Res. 74, 241 (1947)). The pathogen can prevent recognition by the plant by means of structural modifications of the elicitor, masking of the recognition sequence or by competition with another substance for the binding sites on the elicitor or receptor.
Preformed resistance factors form the first defense against colonization by pathogenic organisms. These factors can be morphological factors or else substances of the secondary plant metabolism (phytoanticipins). Morphological factors which prevent colonization are hairy leaves, stomatal density and shape, and the nature of the cuticle and of the cell wall.
Recognition, of the pathogen, by the plant may also lead to the induction of resistance factors, i.e. morphological and physiological resistance responses. Many of these responses are the result of a signal cascade. Signal molecules such as Ca2+, NO, reactive oxygen compounds and phytohormones such as ethylene and jasmonate are involved in the cascades and contribute to the crosslinking of the signal pathways. Resistance responses in which morphological structures in the plant cell are modified are the formation of cell wall appositions (papillae), cork and abscission layers, thyllae and the impregnation of the cell wall. The beginning of penetration by a pathogenic fungus can trigger the formation of papillae. Lignin, callose, suberin and hydroxyproline-rich proteins are deposited at the inner cell wall opposite the potential penetration site and are crosslinked with one another. Callose can be stained by the intercalation of aniline blue. In addition, the papilla formed accumulates phenols, reactive oxygen species and hydrolases (Hückelhoven R. et al., Plant Physiol. 119, 1251 (1999); Assaad F. F. et al., Mol. Biol. of the Cell, 15, 5118 (2004)). The development of papillae leads to a substantially thickened cell wall and may prevent penetration of the pathogenic fungus. Physiological processes which contribute to induced resistance are depolarization of the cell membrane, the oxidative burst, the hypersensitive reaction, the formation of phytoalexins and the expression of pathogenesis-related proteins (PR proteins). One of the first responses to contact with an elicitor is the depolarization of the cell membrane. This results in a pronounced efflux of Cl− and K+ ions, linked with pronounced water loss. It is assumed that depolarization triggers an increased Ca2+ concentration, which is an important signal molecule (Ward J. M. et al., Plant Cell 7, 833 (1995)) and plays a role in the hypersensitive reaction (HR) (Wendehenne a et al., Plant Cell 14, 1937 (2002)). HR in plants is a form of programmed cell death. It allows the plant to stop the fungus even after penetration of the latter by denying it a source of nutrients. The course of HR appears to depend on the combination of plant and pathogen. However, protein biosynthesis, an intact cytoskeleton and salicylic acid appear to be necessary for inducing HR (Heath M., Plant Mol. Biol. 44, 321 (2000)).
A very rapid response to the pathogen is the oxidative burst, the formation of reactive oxygen species, such as the superoxide anion O2−, the hydroxyl radical OH and hydrogen peroxide. These compounds are formed by various oxidases. The hydroxyl radical acts locally, while H2O2 can diffuse via the membranes. Both oxidize polyunsaturated fatty acids and can thus destroy membranes (Grant J. J. et al., Plant Physiol. 124, 21 (2000)). H2O2 is also suspected of performing a function in gene regulation. In addition, the compounds support the defense responses by crosslinking the cell wall components, by increasing lignification and by exerting a toxic effect on pathogens (Garcia-Brugger A. et al., MPMI 19, 711 (2006)). Last but not least, the pathogen attack leads to the expression of genes which code for PR proteins and for phytoalexins. PR proteins are a heterogeneous group of proteins which have a toxic effect on penetrating fungi. The term phytoalexins refers to low-molecular-weight antimicrobially active substances whose synthesis is triggered by biotic or abiotic stress (Prell H. H., Interaktionen von Pflanzen and phytopathogenen Pilzen [Interactions between plants and phytopathogenic fungi], Gustav Fischer Verlag, Jena, 1996; van Loon L. C. et al., Physiol. Mol. Plant Physiol. 55, 85 (1999)). The responses described proceed partly not only when the pathogen interact with a host plant, but also when it reacts with a non-host-plant. Decisive for pathogen defense is the quality of the recognition and the quantity and speed of the resistance response (Thordal-Christensen H., Current Opinion in Plant Biology 6, 351. (2003)).
A plant disease which has become increasingly important in recent times is soybean rust. The disease is caused by the pathogenic rusts Phakopsora pachyrhizi (Sydow) and Phakopsora meibomiae (Arthur). They belong to the class Basidiomycota, order Uredinales, family Phakopsoraceae. The two species are very closely related with one another. The intergenic sequences of their rRNA genes show 80% similarity (Frederick R. D. et al., Phytopathology 92, 217 (2002)). The species are distinguished by morphological characteristics of the teliospores (Ono Y. et al., Mycol. Res. 96, 825 (1992)). Both rusts infect a wide spectrum of host plants. P. pachyrhizi, also referred to as Asian soybean rust, is the more aggressive pathogen on soybeans (Glycine max), and is therefore, at least currently, of great importance for agriculture. P. pachyrhizi is capable of infecting 31 species from 17 families of the Leguminosae under natural conditions and is capable of growing on further 60 species under controlled conditions (Sinclair et al. (eds.), Proceedings of the soybean rust workshop (1995), National Soybean Research Laboratory, Publication No. 1 (1996); Rytter J. L. et al., Plant Dis. 87, 818 (1984)). P. meibomiae has been found in the Caribbean Basin and in Puerto Rico, and has not caused substantial damage as yet.
P. pachyrhizi was originally discovered in Japan in 1902. From there,P. pachyrhizi spread over large parts of Asia and over India and Australia and, finally, reached Africa in 1996. In 2001, the fungus arrived in South America and reached America for the first time in 2004 (Sconyers E.L. et al., at ers.usda.gov/Features/SoyBeanRust/ (2005)). In South America in particular, P. pachyrhizi caused big yield losses of up to 80%. It is estimated that 1.5 million tons of the Brazilian soybean harvest 2005/2006 alone have succumbed to infection with soybean rust. P. pachyrhizi is a hemicyclic rust which forms three types of spores. The formation of teliospores was observed in Asia towards the end of the vegetation period (Yeh C.C. et al., Phytopathology 71, 1111 (1981)). The formation of basidiospores, in contrast, is only known under laboratory conditions. The most important spore form are the uredospores, which are formed over the entire vegetation period and which serve to spread the disease. These spores are formed in large amounts and are capable of spreading over wide distances with the aid of wind and rain. P. pachyrhizi is an obligate biotroph. If a uredospore arrives on a suitable host, it germinates with a single germ tube. At the end of this germ tube, an appressorium develops and rapidly reaches the size of the spore. With the appressorium, the fungus is capable of building up a large pressure and of penetrating the epidermal cells directly with the aid of a penetration hypha. The penetration hypha of P. pachyrhizi grows through the epidermal cell and, once it reaches the intercellular space in the leaf, forms the first septum. It now continues to grow in the leaf as a primary hypha. As early as 24-48 h after the infection, the first haustorial mother cell is divided by a septum, and a sacciform haustorium is formed in a mesophyll cell of the leaf. The cell wall of the mesophyll cell is penetrated, but the plasmalemma is only folded, so that the cell remains alive and can act as a nutrient source. The nutrients travel from the membrane of the live host cell via the extrahaustorial matrix to the haustorium. The epidermal cell which has been penetrated at the beginning turns necrotic shortly after penetration. This manner of infection of a biotrophic pathogen, as is used by P. pachyrhizi, will therefore be referred to as “heminecrotrophic” for the purposes of the description of the present invention. The first uredospores are found only 11-12 days after the infection, and the cycle can start afresh (Koch E. et al., Phytopath, p. 106, 302 (1983)).
The crop plant soybean Glycine max (L.) Merr. belongs to the family Leguminosae, subfamily Papilionoideae, tribe Phaseoleae, genus Glycine Willd. and subgenus soja (Moench). Soybean is planted in more than 35 countries. Some of the most important production areas are located in the United States, China, Korea, Argentina and Brazil. It is considered to be one of the oldest crop plants and was domesticated for the first time in China between the 11th and 17th century (Hymowitz T., Econ. Bot. 24, 408 (1970)). It was introduced into the United States in 1765; the United States are currently one of the largest soya production areas. Wild soybean species can be found in China, Korea, Japan, Taiwan and the former USSR. Morphological, cytological and molecular evidence suggests that G. soja is the ancestor of the cultivated form G. max. Being a subtropical plant, soybeans prefer a mean annual temperature of 5.9-27° C.; they are not frost resistant (OECD, Consensus document on the biology of Gycine max (L.) Merr. (Soybean); Series on harmonization of regulatory oversight in biotechnology No. 15, ENV/JM/MONO(2000)9). Soybeans are currently an important oil and protein source. This extensive use of soya in food production underlines the importance of efficient control of soybean rust.
Soybean plants are infected by P. pachyrhizi by windborne uredospores. The first discernible symptoms are small yellow to reddish-brown lesions on the upper surface of the leaf, which later spread further until all of the leaf finally turns chlorotic and dies. Upon advanced infection, the lesions are found on all of the plant. The first uredia have a diameter of 100-200 μm and are found on the underside of the leaf 10-14 days after the infection; they can produce spores for three to six weeks. Telia are formed subepidermally and mostly occur on the periphery of the lesions. The spores are first yellow to brown and later turn black. The first symptoms are frequently first observed on the older leaves. The rapid development of the disease correlates with the beginning of flowering (R1+) and finally destroys all of the foliage. The fact that most of the photosynthetically active area is destroyed and that water and nutrients are extracted by the fungus leads to reduced productivity of the plant (Sconyers E.L. et al., at ers.usda.gov/Features/SoyBeanRust/ (2005)).
In order to germinate, P. pachyrhizi requires moisture in the form of dew or the like on the upper surface of the leaf. The fungus is encouraged in particular by frequent rain and temperatures of between 15 and 29° C. (Sconyers E.L. et al., at ers.usda.gov/Features/SoyBeanRust/ (2005)). Frequently, the disease starts at discrete locations and subsequently spreads rapidly over the entire field. The fungus is autoecious, i.e. it requires no host alternation for its development, and it can persist readily on its numerous alternative host plants. In the United States, kudzu vine (Pueraria lobata), which originates in Japan, is considered to be a potential host plant on which P, pachyrhizi can overwinter and provide fresh inoculum in the next spring.
P. pachyrhizi can currently be controlled in the field only by means of fungicides. Soybean plants with resistance to the entire spectrum of the isolates are not available. When searching for resistant plants, four dominant genes Rpp1-4, which mediate resistance of soya to P. pachyrhizi, were discovered; however, this resistance is only isolate-specific (Hartwig E. E. et al., Crop Science, 23, 237 (1983); Hartwig E. E., Crop Science 26, 1135 (1986)). Since the resistance was only based on individual genes, it was lost rapidly. Only the Rpp4-mediated resistance has as yet only been broken down under greenhouse conditions (Posada-Buitrago M. L. et al., Fungal Genetics and Biology 42, 949 (2005). The utilization of potential resistance sources from representatives of the perennial subgenus soja is limited (Hartman G. I. et al., Plant Disease 76, 396 (1992)). So far, all crosses have only led to sterile progeny (Singh R. et al., Wendl. Theor. Appl. Genet 74, 391 (1987).
The efficient control of soybean rust with fungicides requires low application into the foliage of the plants, since infection occurs first on the lower leaves. A double treatment has proved to be effective. A disadvantage of the fungicides used is that, as the result of their specific mechanism of action, resistances may develop readily. A potential alternative to the use of fungicides is the use of glyphosate-resistant soybean plants. In a greenhouse experiment, soybean plants were treated with the herbicide three days before inoculation, and a reduction of rust-caused lesions by 46-70% was observed (Feng C. C. P. et al., PNAS 102, 17290 (2005)). Whether this effect will also be retained in field trials remains to be seen.
In recent years, P. pachyrhizi has gained in importance as pest in soybean production. There was therefore a demand in the prior art for developing methods of controlling the fungus. Right now, plant breeding cannot be expected to contribute since the available resistance sources are not accessible. Treatment with fungicides has a limited efficiency and is only effective when the disease is yet to break out. This is why in particular the pathosystem soya/P. pachyrhizi is the method of choice for a recombinant approach. For an approach to be successful, it is initially important to have detailed knowledge of the course of infection and the response of the plant, in particular during the first stages of the infection. Potential candidate genes which confer resistance must be identified, and their effect in the interaction between plant and pathogen must be characterized. The stable transformation of plants is very time-consuming and costly, which is why transient transformation, which makes possible a characterization of the plant/pathogen interaction at the cell level, is preferred. The method of choice here is the transient transformation of leaves with the gene gun. In plants, non-host-resistance is particularly effective and durable and based on the fact that quantity and speed of the resistance response is increased in comparison with the compatibility response. Thus, the characterization of decisive genes in non-host-resistance can serve to identify potential candidate genes for the generation of resistant plants. Thus, to also study the response of a non-host-plant to soya rust, the system barley (Hordeum vulgare)/P. pachyrhizi was chosen since barley is a model plant which has been described in detail. Moreover, the pathosystem barley/with Blumeria graminis f.sp. hordei (Bgh) and with Blumeria graminis f.sp. tritici (Bgt), which is incompatible, has been studied in great detail. Although Bgh and P. pachyrhizi differ in some respects, they share at least phenotypically some steps at the beginning of the infection process. Thus, the analysis in barley can provide information on the mechanisms of the non-host-resistance to P. pachyrhizi in comparison with the host resistance of barley. Thus, candidate genes were tested in transiently transformed barley leaves for their effect in the resistance to P. pachyrhizi. The transient transformation with the gene gun provides a method of studying, in the pathosystem barley/P. pachyrhizi, a series of genes which may be involved in the resistance response.
Programmed cell death (PCD) is an important process in the development and stress response of plants and animals. Some morphological and biochemical changes in the cell, such as chromatin condensation, shrinking of the cytoplasm and DNA fragmentation, appear to be shared by plants and animals (Lam E. et al., Nature 411, 848 (2001)). A clear distinction between PCD and HR, which is caused by biotic or abiotic stress, is not possible (Heath M., Plant Mol. Biol. 44, 321 (2000)). One activator of PCD in animals is BAX. BAX develops channels in the outer mitochondrial membrane and causes the release of cytochrome c. This triggers a caspase cascade, and thus the proteolysis of proteins which the cell needs to survive (Green D. R. et al, Science 281, 1309 (1998). The overexpression of BAX in tobacco (N. tabacum, Lacomme C. et al., Proc. Nat. Acad. Sci. USA 96, 7956 (1999)) and Arabidopsis thaliana (Kawai-Yamada M. et al., Plant Cell 16, 21 (2004)) causes PCD and thus suggests similar mechanisms in plants and animals. However, no BAX homologs have been identified in plants. However, a BAX-antagonistic regulator of PCD, the Bax inhibitor-1 (BI-1), is conserved in plants, animals and other organisms such as yeast. Similar proteins have been identified since in A. thaliana, H. vulgare, Brassica napus, Brassica oleracea, Oryza sativa and N. tabacum (Hückelhoven R., Apoptosis 9, 299 (2004)). In experiments with BI-1/GFP fusion proteins, a localization of BI-1 in the membrane of the endoplasmic reticulum (ER) and the nuclear membrane has been observed (Eichmann R. et al., Mol. Plant Microbe Interact. 17, 484 (2004)). The protein has a size of 25-27 kDa and has 6-7 transmembrane domains. The C-terminal end, which is probably located in the cytoplasm (Bolduc N. et al., Planta 216, 377 (2003)), is essential for the function of BI-1 (Kawai-Yamada et al., Plant Cell 16, 21 (2004)). It is possible that the transmembrane domains form an ion channel (Bolduc N. et al., Planta 216, 377 (2003)). Thus, BI-1 might have a function in regulating the cytosolic Ca2+ level and/or the redox state of the cell as the result of the ER's storage function for Ca2+ (Xu Q. et al., Mol. Cell 18, 1084 (1998); Balduc N. et al., FEBS Lett 532, 111 (2003); Hückelhoven R. et al., Proc. Natl. Aced Sci. USA 29, 5555 (2003); Matsumura H. et al., Plant J. 33, 425 (2003)). In animal cells, there is no direct physical interaction between BI-1 and Bax. However, BI00-1 interacts with other PCD regulators (Xu Q. et al., Mol. Cell 18, 1084 (1998)). It is probable that BI-1, in plants, also interacts with other PCD regulators, thus influencing the resistance responses. In Arabidopsis, BI-1 is capable of suppressing BAX and the H2O2 have induced PCD (Baek et al., Plant Mol. Biol. 56, 15 (2004); Kawai-Yamada M. et al., Plant Cell 16, 21 (2004)). Therefore, it probably regulates the processes at a level lower than the oxidative stress response (Kawai-Yamada M. et al., Plant Cell 16, 21 (2004)).
The expression of BI-1 is induced by biotic and abiotic stress such as attack by pathogens or wounding, but also in aging tissues (Balduc N. et al., FEBS Lett. 532, 111 (2003); Hückelhoven R., Apoptosis 9, 299 (2004)). In Arabidopsis, the mRNA levels of BI-1 are increased after heat shock (Watanabe N. et al., Plant J. 45, 884 (2006)). BI-1 expression is induced in tomato (Lycopersicum esculentum) by H2O2, and in Arabidopsis by H2O2 and salicylic acid (Hückelhoven R., Apoptosis 9, 299 (2004); Kawai-Yamada M. et al., Plant Cell 16, 21 (2004)). This suggests that BI-1 has a function in pathogen defense, because this is where both substances play an important role (Prell H. H., Interaktionen von Pflanzen and phytopathogenen Pilzen [Interactions between plants and phytopathogenic fungi], Gustav Fischer Verlag, Jena, 1996).
Accordingly, the infection of barley with Bgh or Bgt triggers an increased expression of BI-1 (Hückelhoven R. et al., Plant Mol. Biol. 47, 739 (2001); Eichmann R. et al., Mol. Plant Microbe Interact. 17, 484 (2004)). In rice, the expression is biphasic after infection with M. grisea. It is first slightly increased, but is reduced 12 hours after the infection only to rise again (Matsumura H. et al., Plant J. 33, 425 (2003)), The increased expression of BI-1 in Arabidopsis cells after treatment with fumonisin B1 (Watanabe N. et al., Plant J. 45, 884 (2006)), and the reduction of the BI-1 expression after the treatment of rice cells with M. grisea elicitor extract, demonstrates that the expression patterns can differ greatly, depending on the inducing factor and on the plant. The expression patterns suggest a role of BI-1 in the regulation of the stress-induced PCD or HR and in the resistance response to pathogens. The influence on the HR can increase the resistance of a plant, especially if the pathogens are pertotrophic or hemibiotrophic fungi. Overexpression of BI-1 in carrots (Daucus carota ssp. sativa) leads to resistance of the plants to Botrytis cinerea Omani J. et al., Mol. Plant Physiol. in press). In tomatoes, the expression of the PCD inhibitor p35 protects against Alternaria alternata, Colletotrichum coccodes and Pseudomonas syringae (Lincoln J. E. et al., Proc. Nat. Acad. Sci. USA 99, 15217 (2002)).
Against this background, there was a continuous demand in the prior art for crop plants with an increased resistance to pathogens. Only few approaches exist which confer, to plants, a resistance to a broader spectrum of pathogens, especially fungal pathogens. Systemic acquired resistance (SAR)—a defense mechanism in various plant/pathogen interactions—can be conferred by application of endogenous messenger substances such as jasmonate (JA) or salicylic acid (SA) (Ward J. M., et al., Plant Cell 3, 1085 (1991); Uknes et al., 4(6), 645 (1992)). Similar effects can also be brought about by synthetic compounds such as 2,6-dichloroisonicotinic acid (DCINA) or benzo(1,2,3)thiadiazole-7-thiocarboxylic acid S-methyl ester (BTH; Bion®) (Friedrich et al., Plant J. 10(1), 61 (1996); Lawton et al., Plant J. 10, 71 (1996)). Also, expression of “pathogenesis-related” (PR) proteins, which has been upregulated in the context of SAR, may partly bring about resistance to pathogens.
In barley, the Mlo locus has been described as a negative regulator of pathogen defense. The loss, or loss of function, of the Mlo gene brings about an increased, race-unspecific resistance to a large number of mildew isolates (Büschges R. et al., Cell 88, 695 1997); Jorgensen J. H., Euphytica 26, 55 (1997); Lyngkjaer M. F. et al., Plant Pathol 44, 786 (1995)).
The Mlo gene has been described (Büschges R. et al., Cell 88, 695 (1997); WO 98/04586; Schulze-Lefert P. et al., Trends Plant Sci. 5, 343 (2000)). Various Mlo homologs from other cereal species have been isolated. Methods using these genes for obtaining pathogen resistance have been described (WO 98/04586; WO 00/01722; WO 99/47552). The disadvantage is that Mlo-deficient plants also initiate the abovementioned defense mechanism in the absence of a pathogen, which manifests itself in the spontaneous dying of plant cells (Wolter M. et al., Mol. Gen. Genet. 239, 122 (1993)). As the result, mlo-resistant plants suffer a yield loss of up to 5% (Jörgensen J.H. Euphytica 63, 141 (1992)). The spontaneous dying of the leaf cells furthermore brings about a disadvantageous hypersusceptibility to necrotrophic and hemibiotrophic pathogens such as Magnaporthe grisea (M. grisea) or Cochliobolus sativus (Bipolaris sorokiniana) (Jarosch B. et al., Mol Plant Microbe Interact. 12, 508 (1999); Kumar J. et al., Phytopathology 91, 127 (2001)).
Apoptosis, also referred to as programmed cell death, is an essential mechanism for maintaining tissue homoeostasis, and, as such, counteracts cell division as a negatively-regulating mechanism. In the multi-celled organism, apoptosis is a natural component of ontogenesis, and involved, inter alia, in organ development and the removal of senescent, infected or mutated cells. As the result of apoptosis, undesired cells are eliminated in an efficient manner. Interference with, or inhibition of, apoptosis contributes to the pathogenesis of a variety of diseases, among which carcinogenesis. The main effectors of apoptosis are aspartate-specific cysteine proteases, which are known by the name of caspases. They can generally be activated by at least two apoptotic signal pathways: firstly by the activation of the
TNF (tumor necrosis factor) receptor family; secondly, the mitochondria play a central role. Activation of the mitochondrial apoptosis signal pathway is regulated by proteins of the Bcl-2 family. This protein family consists of antiapoptotic and proapoptotic proteins such as, for example, Bax. In the case of an apoptotic stimulus, the Bax protein undergoes an allosteric conformation change, which leads to the anchoring of the protein in the external mitochondrial membrane, and to its oligomerization. As the result of these oligomers, proapoptotic molecules are released from the mitochondria into the cytosol and bring about an apoptotic signal cascade and, ultimately, the degradation of specific cellular substrates, resulting in cell death. The Bax inhibitor-1 (BI1) was isolated via its property of inhibiting the proapoptotic effect of BAX (Xu Q. et al., Mol Cell 1(3), 337 (1998)). BI1 is a highly conserved protein. It is found predominantly as an integral constituent of intracellular membranes. BI1 interacts with bcl-2 and bcl-xl. The overexpression of BI1 in mammalian cells suppresses the proapoptotic effect of BAX, etoposid and staurosporin, but not of Fas antigen (Roth W. et al., Nat. Med. 8, 216 (2002)). The inhibition of BI1 by antisense RNA, in contrast, induces apoptosis (Xu Q. et al., Mol Cell 1(3), 337 (1998)). The first plant homologs of BI1 have been isolated from rice and Arabidopsis (Kawai et al., FEBS Lett 464, 143 (1999); Sanchez et al., Plant J. 21, 393 (2000)). These plant proteins suppress the BAX-induced cell death in yeast. The amino acid sequence homology with human BI1 is approximately 45%. In recombinant plants, the Arabidopsis homolog AtBI1 is capable of suppressing the proapoptotic effect of murine BAX (Kawai-Yamada M. et al., Proc. Natl. Acad. Sci. USA 98(21), 12295 (2001)). The rice (Oryza sativa) BI1 homolog OsBI1 is expressed in all plant tissues (Kawai et al., FEBS Lett 464, 143(1999)). Furthermore described are BI1 genes from barley (Hordeum vulgare; GenBank Acc.-No.: AJ290421), rice (GenBank Acc.-No.: AB025926), Arabidopsis (GenBank Acc.-No.: AB025927), tobacco (GenBank Acc.-No.: AF390556) and oilseed rape (GenBank Acc.-No.: AF390555, Bolduc N. et al., Planta 216, 377-386 (2003)). The expression of BI1 in barley is upregulated as the result of infection with mildew (Hückelhoven R. et al., Plant Mol. Biol. 47(6), 739 (2001)).
WO 00/26391 describes the overexpression, in plants, of the antiapoptotic genes Ced-9 from C. elegans, sfIAP from Spodoptera frugiperda, bcl-2 from humans and bcl-xl from chicken for increasing the resistance to necrotrophic or hemibiotrophic fungi. Plant BI1 homologs are not disclosed. Expression is under the control of constitutive promoters. Furthermore described is the expression of a BI1 protein from Arabidopsis under the strong constitutive 35S CaMV promoter in rice cells and a hereby-induced resistance to cell-death-inducing substances from Magnaporthe grisea (Matsumura H. et al., Plant J. 33, 425 (2003)).
Originally, the prior art described that constitutive expression of an inhibitor of the programmed cell death in plants can bring about resistance to necrotrophic fungi.
However, the person skilled in the art was faced in particular with the problem of providing methods for the pathogen defense in plants, in particular against biotrophic pathogens.
Surprisingly, the problem is solved by the inventive methods, peptide sequences, nucleic acid sequences, expression cassettes, vectors and organisms defined in the main claims, using a BI1 protein. The dependent claims define specific, especially preferred use forms of the present invention.
The roll of BI-1 has been tested in three independent experiments in the transient transformation system. In the control, 53% (averaged over the experiments) of the transformed cells which interacted with P. pachyrhizi were penetrated, while only 37% of the BI-1 transformed cells were penetrated (FIG. 8; Table FIG. 10). The data are based on three independent experiments. Barley leaves were transformed with the reporter gene construct pGY1-GFP and the blank vector in order to act as controls. The penetration rate in the BI-1 transformed cells differs significantly from the WT (P<0.05). After the evaluation, the cells which have been transiently transformed with BI-1 therefore surprisingly show a significantly increased penetration resistance to P. pachyrhizi (P<0.05; FIG. 8; Table FIG. 10). A noteworthy aspect of the observation under the microscope was that the BI-1-forming cells had a markedly more vital appearance than cells, which expressed GFP or ADF3.
Thus, even the transient overexpression of the cell death inhibitor BI-1 in barley revealed, surprisingly, the trend to an increased resistance of the plant cells to penetration by soybean rust. To confirm this, the interaction between transgenic barley plants cv. “Golden Promise” (GP) which contain a GFP-BI-1 overexpression construct was studied under the microscope in comparison with the wild type (WT) of this variety. To generate the transgenic plants, a GFP-BI-1 fusion under the control of the constitutive CaMV 35S promoter was used, thus ensuring sufficient expression of the protein.
In preliminary experiments, leaves of the WT cv. “Golden Promise” and of the transgenic barley line (cv. “Golden Promise”) #6(1)E8L1(T1)′ were inoculated with P. pachyrhizi and, 24 hours after the inoculation, fixed in destaining solution. After destaining was complete, the leaves were stained with aniline blue. Aniline blue intercalates into the structure of callose and thus preferentially stains papillae where callose undergoes accumulation and crosslinking with other polymeric substances. Cells which, as the result of a hypersensitive response (HR), have undergone a similar process as apoptosis in mammalian cells, will, after staining with aniline blue, also show a light fluorescence. The number of spores, the germinated spores with germ tubes which had already formed an appressorium and, as cell response, the appressoria with underlying papillae and the HR were counted on the inoculated barley leaves. Larger spore agglomerations where an assignation of the appressoria to the spores was no longer possible were not included. As far as possible, at least 100 spores with appressoria were counted per leaf. Even in this experiment, a significantly increased formation of papillae and a significantly decreased HR of the cells was observed in the transgenic line (P<0.01; Table FIG. 11A/B, FIG. 12).
Both Bgh (Hückelhoven R., FEMS Microbiol. Letters 245, 9(2005)) and P. pachyrhizi (Koch E. et al., Phytopath, p. 106, 302 (1983)) are biotrophic pathogens. An HR of the infected cells can therefore stop the development of the fungi since it deprives them from their food source. If the HR is prevented, the cells may become more sensitive to infection by biotrophic pathogens. Despite this, barley cells which have been transiently transformed with a BI-1 overexpression construct surprisingly show an increased resistance to penetration by P. pachyrhizi. This is surprising because an increased sensitivity to the biotrophic fungus would have been expected However, the HR is not the only resistance reaction in the resistance of barley and wheat to the incompatible pathogen P. pachyrhizi, and perhaps not the decisive one Thus, the defense of wheat against P. pachyrhizi is papille formation (Hoppe H. H. et al., Pro. Intern. Congress of SABRAO (Bangkok 1985) 1986). Barley responds with papilla formation and with HR of the infected cells, depending on the variety. Moreover, barley with the mlo5 allele responds with papilla increased papilla formation. The defense of barley to Bgt is also affected by papilla formation, but mostly by an HR of infected cells (Hückelhoven R. et al., Mol. Plant Pathol. 2, 199 (2001). In contrast to P. pachyrhizi, the transient overexpression of BI-1 in barley leads to an increased penetration rate of the cells by Bgt (Eichmann R. et al., Mol. Plant Microbe Interact. 17, 484 (2004)). Likewise, barley plants with the mlo5 allele, which confers broad resistance to Bgh demonstrate, in the case of transient overexpression of BI-1, greater sensitivity of the cells to penetration by Bgh (Hückelhoven R. et al., Proc. Natl. Acad. Sci. USA, 29, 5555 (2003). Although the resistance of barley with the mlo5 allele is based not on an HR of the infected cells, but on a more efficient accumulation of antimicrobial compounds, H2O2 and an increased formation of papillae, the inhibition of the HR also appears to affect the other resistance responses (Hückelhoven R. et al., Plant Physiol. 119, 1251 (1999)). Without causing limitation by theory, these observations of a reduced penetration resistance as the result of inhibition of HR suggests that crosslinked regulation of the resistance responses. The suspicion that the resistance responses are subject to crosslinked regulation is supported by the microscopic analysis of transgenic barley plants which have been inoculated with P. pachyrhizi. These barley plants contain a BI-1 overexpression construct and respond to infection with increased papilla formation. While only 16-26% of the infectcd cells show HR on the leaves of the transgenic plants, HR was observed in approximately 50% of the infected cells on the WT leaves. Consequently, in the barley variety “Golden Promise”, the HR of the cells also appears to be an important resistance mechanism against P. pachyrhizi, which is a biotroph, although P. pachyrhizi does not utilize the epidermal cells as a food source, at least not in soybean, but only forms haustoria in the mesophyll (Koch E. et al., Phytopath, p. 106, 302 (1983)). In general, however, the fungus did not reach this stage on the non-host plant barley. Inhibition of the HR makes it possible for Bgt to penetrate the cells, and the fungus can establish itself successfully (Eichmann R. et al., Mol. Plant Microbe Interact. 17, 484 (2004)). Further fungus-specific factors appear to be necessary for this process. It appears that these specific factors of Bgt are capable of suppressing either the alternative resistance response of the plant cell or the recognition by the latter. These factors are probably absent in P. pachyrhizi. Thus, the plant cell is perhaps capable of recognizing P. pachyrhizi and, since BI-1 suppresses the HR, capable of inducing an alternative resistance response, papilla formation. Recognition of P. pachyrhizi by the plant is supported by the observation of increased papilla formation in barley with the mlo5 allele. However, the reason why the plant is capable of recognizing the pathogen P. pachyrhizi, which is specialized to host plants of a completely different order, remains unexplained (Sinclair J. B. et al. (eds.), Proceedings of the soybean rust workshop (1995), National Soybean Research Laboratory, Publication No. 1 (1996)). The “recognition feature” of P. pachyrhizi might be an unspecific elicitor (‘pathogenesis-associated molecular patterns’ PAMP) such as the INF1 from P. infestans, which triggers an HR in Nicotiana sp. (Kamoun S. et al., Plant Cell 10, 1413 (1998)). PAMPs are recognized by the plant as foreign molecules and trigger a resistance response. Not all leaves of the transgenic plant show increased papilla formation, although the BI-1 gene construct has been identified in them by means of PCR. This might be the consequence of an unfavorable insertion type of the construct, which prevents an effective expression of BI-1 and/or leads to antagonistic effects by other genes. Since the identification was performed at the DNA level, no comments can be made on the expression of BI-1. It must also be borne in mind that even minor damage to the seeds or the leaves can have a decisive effect, or prevent, the development of the plant. Thus, some seeds of the line #6(2)E15L7P2 (T2) did not germinate.